The ability to genetically engineer monocots, including cereal crops, to improve their performance and pest-resistance, or to enhance alternative uses is of great importance. Genetic improvement of various crop species by genetic engineering has sometimes been hindered because techniques for in vitro culture, transformation, and regeneration of amenable cultivars are less effective with recalcitrant commercial cultivars.
In particular, the development of stable transformation technologies for cereal plants is largely dependent on the availability of efficient methods for inducing and maintaining large numbers of undifferentiated plant cells in culture.
Virtually all current genetic engineering technologies require that genes be delivered to cells grown in vitro. Many published methods for generating fertile transformed plants from cereals (e.g. rice, wheat, maize, oat, sorghum, triticale, barley and rye) utilize as initial explants the immature scutellum of the embryo or microspores directly or tissue derived from immature embryos or microspores. From these initial explants, cellular proliferation occurs. Maintenance and regeneration of these proliferating cells is required for almost all stages of genetic transformation methods. The cells can then be stably transformed with the gene or genes of interest and the transformed cells can be selected. After selection or screening for transformants, plants are regenerated.
Most transformation protocols require that the target tissue undergo embryogenesis, which may include de-differentiation of a single original transformed cell before the sustained cell divisions that give rise to an embryo consisting mostly or entirely of cells that contain the introduced DNA. De-differentiation during in vitro culturing introduces stresses on the genome, causing modifications of the genome that are associated with somaclonal variation, including DNA methylation, point mutations, deletions, insertions, and the generation of gross cytogenetic abnormalities. These genomic modifications lead to subsequent phenotypic abnormalities and performance losses and may contribute to other problems
Transformation methods using excised shoot apices have been previously described (see, for example, U.S. Pat. No. 5,164,310 to Smith et al.; Zhong et al. 1996, both of which are herein incorporated by reference). However, these methods have not proven to be effective for maize that include commercially important elite inbreds. There is a need, therefore, for improved methods for plant transformation and regeneration, particularly for use with maize elite inbreds.